Objective Currently, there is no commercially available quantitative detection kit for the main Felis domestic allergen (Fel d 1) in China. To establish a rapid detection method for Fel d 1, this study aims to prepare monoclonal antibodies against Fel d 1 protein. Methods The codon preference of Escherichia coli was utilized to optimize and synthesize the Fel d 1 gene. The prokaryotic expression plasmid pET-28a-Fel d 1 was constructed and used to express and purify the recombinant Fel d 1 protein. Subsequently, the recombinant protein was immunized into BALB/c mice and monoclonal antibodies (mAbs) were prepared by the hybridoma technique. An indirect ELISA was established using the recombinant Fel d 1 as the coating antigen, and hybridoma cell lines were screened for positive clones. The specificity and antigenic epitopes of the mAbs were confirmed by Western blot analysis. Finally, the selected hybridoma cells were injected into the peritoneal cavities of BALB/c mice for large-scale monoclonal antibody production. Results The recombinant plasmid pET-28a-Fel d 1 was successfully constructed, and soluble Fel d 1 protein was obtained after optimizing the expression conditions. Western blot and antibody titer assays confirmed the successful isolation of two hybridoma cell lines, 7D11 and 5H4, which stably secreted mAbs specific to Fel d 1. Antibody characterization revealed that the 5H4 mAb was of the IgG2a subtype and could recognize the amino acid region 105-163 of Fel d 1, while the 7D11 mAb was the IgG1 subtype and could recognize the amino acid region 1-59. Conclusion The high-purity recombinant Fel d 1 protein produced in this study provides a promising alternative for clinical immunotherapy of cat allergies. Furthermore, the monoclonal antibody prepared in this experiment lays a material foundation for the in-depth study of the biological function of Fel d 1 and the development of ELISA detection.
Animals ; Antibodies, Monoclonal/biosynthesis* ; Mice, Inbred BALB C ; Cats ; Mice ; Allergens/genetics* ; Glycoproteins/genetics* ; Enzyme-Linked Immunosorbent Assay ; Hybridomas/immunology* ; Recombinant Proteins/genetics* ; Female ; Antibody Specificity

Objective To prepare CD318-specific monoclonal antibodies and evaluate their specificity, affinity, and application in immunological detection, laying the foundation for the development of CD318-targeted antibody drugs. MethodsCD318 protein was expressed and purified, and was used as an antigen to immunize mice, then mice with higher antiserum titers were screened. We prepared CD318-specific monoclonal antibodies through cell fusion and monoclonal screening, and the specificity, affinity, and application of the obtained monoclonal antibodies in immunological assays were evaluated. Then we constructed a CD318/CD3-targeting bispecific antibody and assessed its impact on T-cell cytotoxicity. Results Thirteen monoclonal antibodies were successfully generated, with the hybridoma clone 13-8-G2 exhibiting the highest titer, strongest specificity, and broadest applicability. The antibody was identified as an IgG1 isotype with a kappa light chain. The variable region of the light chain measured 318 bp, while the heavy chain variable region was 357 bp, yielding an affinity constant of approximately 7.68×10⁹. The specificity of CD318 was confirmed using flow cytometry and immunofluorescence assays. Additionally, a CD318/CD3-targeting bispecific antibody was constructed using the variable regions of this CD318 monoclonal antibody, which demonstrated enhanced T-cell cytotoxicity. Conclusion High-affinity and highly specific CD318 monoclonal antibodies were successfully prepared, laying a foundation for the development of therapeutic antibodies targeting CD318.
Animals ; Antibodies, Monoclonal/biosynthesis* ; Mice ; Antibodies, Bispecific/immunology* ; Humans ; Mice, Inbred BALB C ; Antibody Specificity/immunology* ; CD3 Complex/immunology* ; Antigens, CD/genetics* ; T-Lymphocytes/immunology* ; Hybridomas/immunology* ; Female

The global outbreak of monkeypox in 2022 has aroused widespread concern in public health. To date, the prevention and treatment of monkeypox has mainly relied on smallpox vaccines and drugs. This study aims to screen and obtain therapeutic antibodies with high affinity, neutralizing activity, and protective effects, and provide candidate molecules for the development of specific therapeutic antibodies against monkeypox. Therefore, humanized mice were immunized to screen for antibodies against the envelope protein of the mpox virus. Two M1R-specific antibodies, 12G5 and 12H6, were obtained, with the affinity of 0.095 nmol/L and 0.089 nmol/L, respectively. The 50% reduction of the plaque counts (PRNT₅₀) of 12G5 and 12H6 was (1.821±1.766) μg/mL and (17.605±2.383) μg/mL, respectively. The two antibodies targeted two binding epitopes of M1R. Moreover, 12H6 could protect 60% of mice from death following the vaccinia virus challenge. This study provides research materials for subsequent in-depth studies on the immunoprotection of mpox virus and potential therapeutic strategies.
Animals ; Mice ; Antibodies, Viral/immunology* ; Monkeypox virus/immunology* ; Mpox, Monkeypox/immunology* ; Antibodies, Neutralizing/immunology* ; Viral Envelope Proteins/immunology* ; Humans ; Antibodies, Monoclonal/biosynthesis* ; Female

Hemoglobin A1c (HbA1c) has a unique structure that makes monoclonal antibody (mAb) preparation challenging. This study aims to develop a method for preparing HbA1c mAbs and establish a fluorescent immunochromatographic assay (FICA) for rapid detection of HbA1c. Three glycosylated peptides were synthesized and used to prepare complete antigens, which were identified by dot enzyme-linked immunosorbent assay (Dot-ELISA) and ultraviolet absorption spectroscopy. The complete antigens and natural HbA1c were used for cross-immunization of mice, and the optimal complete antigen was selected. The mouse with the highest serum titer was chosen for mAb preparation. The purity and specificity of the mAbs were verified, and a FICA method was developed. The optimal complete antigen, with a titer of 1:512 000, was successfully prepared and selected. Fusion with splenocytes resulted in four specific HbA1c antibodies (purity > 90%). The best antibody exhibited a binding constant (Ka) of 1.67×10¹⁰ L/mol with the antigen. Based on this antibody, a FICA method was successfully established, capable of producing results within 15 min. The method demonstrated a good linear range (3%-13% HbA1c, y=0.071 3x+0.005 6, R²=0.993 7), recovery rates of 98%-102%, precision < 10.00%, and no nonspecific reactions. Clinical testing of 210 samples showed positive agreement of 96.36%, negative agreement of 97.00%, and overall agreement of 96.68%. The receiver operating characteristic (ROC) curve analysis yielded an area under curve (AUC) of 0.980 9 [95% confidence interval (CI): 0.961 0-1.000 0], with high consistency verified in multicenter studies. We successfully developed a key technique for preparing HbA1c monoclonal antibodies and established a FICA method for rapid detection of HbA1c. It will provide an efficient and convenient detection method for the early diagnosis and long-term management of diabetes and its complications.
Antibodies, Monoclonal/biosynthesis* ; Animals ; Mice ; Glycated Hemoglobin/immunology* ; Mice, Inbred BALB C ; Humans ; Antibody Specificity ; Chromatography, Affinity/methods* ; Enzyme-Linked Immunosorbent Assay/methods* ; Female

Bispecific antibody drugs have enhanced efficacy and safety compared with therapeutic monoclonal antibody drugs, gradually becoming a new generation of effective therapies. With the development of genetic engineering and the maturity of the industry, increasing studies have been carried out on bispecific and multi-specific antibody drugs, and the application indications of these drugs are expanding, which lays a foundation for satisfying the clinical needs and creating clinical values. This paper provides a comprehensive review of the development stages, indications, target combinations, structural formats, and mechanisms of bispecific and multi-specific antibody drugs. It also discusses key points and strategies for differentiated drug discovery, facilitating the rapid development of innovative therapies, providing theoretical support for the clinical translation of these drugs, and offering more precise and effective treatment options for clinical practice.
Antibodies, Bispecific/biosynthesis* ; Humans ; Drug Discovery ; Antibodies, Monoclonal/biosynthesis*

Foodborne pathogens are one of the major factors leading to food safety issues, causing harm to the national economy and people's livelihood. Achieving rapid detection of foodborne pathogens is currently a key strategy for preventing and controlling foodborne diseases. Antibodies naturally possess high specificity and sensitivity, serving as preferred tools for specific recognition of foodborne pathogens. We list the main methods for detecting foodborne pathogens, introduce the evolution and development of polyclonal antibodies, monoclonal antibodies, and genetically engineered antibodies, and review the application of different antibody technologies in the rapid detection of foodborne pathogens. Furthermore, we recognize that the combination of antibody technology and other foodborne pathogen detection methods is currently a reliable means to improve detection performance. Finally, we elaborate on the existing limitations of different antibodies and summarize the current research status and potential issues, aiming to provide a theoretical basis and practical ideas for the development of rapid detection of foodborne pathogens.
Foodborne Diseases/prevention & control* ; Food Microbiology ; Antibodies, Monoclonal/biosynthesis* ; Antibodies/immunology* ; Humans ; Food Contamination/analysis*

The voltage-gated Na channel subtype Nav1.7 is important for pain and itch in rodents and humans. We previously showed that a Nav1.7-targeting monoclonal antibody (SVmab) reduces Na currents and pain and itch responses in mice. Here, we investigated whether recombinant SVmab (rSVmab) binds to and blocks Nav1.7 similar to SVmab. ELISA tests revealed that SVmab was capable of binding to Nav1.7-expressing HEK293 cells, mouse DRG neurons, human nerve tissue, and the voltage-sensor domain II of Nav1.7. In contrast, rSVmab showed no or weak binding to Nav1.7 in these tests. Patch-clamp recordings showed that SVmab, but not rSVmab, markedly inhibited Na currents in Nav1.7-expressing HEK293 cells. Notably, electrical field stimulation increased the blocking activity of SVmab and rSVmab in Nav1.7-expressing HEK293 cells. SVmab was more effective than rSVmab in inhibiting paclitaxel-induced mechanical allodynia. SVmab also bound to human DRG neurons and inhibited their Na currents. Finally, potential reasons for the differential efficacy of SVmab and rSVmab and future directions are discussed.
Animals ; Antibodies, Monoclonal ; therapeutic use ; Biotin ; metabolism ; Cells, Cultured ; Disease Models, Animal ; Female ; Ganglia, Spinal ; cytology ; HEK293 Cells ; Humans ; Hybridomas ; chemistry ; Hyperalgesia ; drug therapy ; Male ; Mice ; Mice, Inbred C57BL ; NAV1.5 Voltage-Gated Sodium Channel ; metabolism ; NAV1.7 Voltage-Gated Sodium Channel ; chemistry ; immunology ; metabolism ; Neuralgia ; drug therapy ; metabolism ; Protein Binding ; drug effects ; Recombinant Proteins ; biosynthesis ; therapeutic use ; Sensory Receptor Cells ; drug effects ; physiology

In order to develop monoclonal antibodies (McAbs) against the gp90 protein of reticuloendotheliosis virus (REV), the His-tagged gp90 protein of REV was used to immunize BALB/c mice. Hybridomas were generated by fusing mouse myeloma cells SP2/0 with the splenocytes from the immunized mice. After screening and 3 rounds of cloning process, 3 hybridomas (3G5-B8, 3G5-A10 and 1G12) that stably secreted McAbs against the REV-gp90 were obtained. The isotypes of the McAbs were determined to be IgG1, IgG1 and IgG2b. The McAbs specifically bound to gp90 in REV-infected DF-1 cells, as demonstrated by Western blotting and indirect immunofluorescence assay. The recognition regions on gp90 that were recognized by 3G5-B8/3G5-A10 and 1G12 were located between amino acids 200 to 245 and 230 to 235, respectively, as demonstrated by Western blotting analysis. These McAbs will be useful in the diagnosis and pathogenesis study of REV.
Animals ; Antibodies, Monoclonal ; biosynthesis ; Antibody Specificity ; Blotting, Western ; Epitope Mapping ; Hybridomas ; Immunoglobulin G ; biosynthesis ; Mice ; Mice, Inbred BALB C ; Reticuloendotheliosis virus ; immunology ; Viral Envelope Proteins ; immunology

OBJECTIVETo prepare rabbit monoclonal antibody (RabMab) against guanosine 3', 5'-cyclic monophosphate (cGMP) and to develop a competitive ELISA for the detection of cGMP.

METHODSNew Zealand white rabbits were immunized with synthesized cGMP-keyhole limpet hemoeyanin (cGMP-KLH) to prepared a RabMAb with monoclonal antibody technique of Epitomics. A competitive ELISA kit was produced with cGMP RabMAb. The specificity, the precision and the recoveries of the method were determined.

RESULTSThe RabMAb with high sensitivity towards cGMP were prepared with an antibody timer of 3.1 ng/mL and 50% inhibitive concentration (IC50) of 12.57 ng/mL. The cGMP RabMAb had 33% cross-reactivity to inosine 3', 5'-cyclic monophosphate (cIMP) and little or no cross-reactivity to other compounds. A competitive ELISA was developed for detection of cGMP. The range of detection was 0~120 ng/mL with a minimal limit of 1.95 ng/mL. The recovery of assay was 89%~103%. The inter-assay and intra-assay coefficient variations were below 11.68% and 13.85%, respectively.

CONCLUSIONThe RabMab against cGMP with high affinity and high specificity has been generated successfully, and a competitive ELISA for detection of cGMP has been developed with the prepared cGMP RabMAb.

Infectious bursal disease virus (IBDV) VP4 plays an important role in immunosuppression of host. In order to develop monoclonal antibodies (McAbs) against VP4, we vaccinated BALB/c mice with His-VP4, screened and subcloned positive clones. We established 4 hybridoma cell lines that stably secreted McAbs against VP4 and named these cell lines 3B3, 3H11, 4C8 and 4G6, respectively. We tested the dissociation constant (Kd) of these McAbs, and found that their K(d)s were 4.61 x 10(-11), 1.71 x 10(-10), 4.26 x 10(-11), 5.02 x 10(-11), respectively. The isotypes of these McAbs were determined to be IgG1, IgG1, IgG2b and IgG1. These McAbs specifically bound to VP4 in IBDV infected DF-1 cells as demonstrated by Western blotting analysis and fluorescence antibody assay. These McAbs would help to detect IBDV infection and to analyze the biological activities of IBDV VP4.
Animals ; Antibodies, Monoclonal ; biosynthesis ; Blotting, Western ; Cell Line ; Fluorescent Antibody Technique ; Hybridomas ; Infectious bursal disease virus ; Mice ; Mice, Inbred BALB C ; Viral Structural Proteins ; immunology